Mitochondrial ion channels as oncological targets

Mitochondrial ion channels as oncological targets L Leanza 1 , M Zoratti 2 , E Gulbins 3 and I Szabo 1 M itochondria, the key bioenergetic intracellular organelles, harbor a number of proteins with proven or hypothetical ion channel functions. Growing evidence points to the important contribution of these channels to the regulation of mitochondrial function, such as ion homeostasis imbalances profoundly affecting energy transducing processes, reactive oxygen species production and mitochondrial integrity. Given the central role of mitochondria in apoptosis, their ion channels with the potential to compromise mitochondrial function have become promising targets for the treatment of malignancies. Importantly, in vivo evidence demonstrates the involvement of the proton-transporting uncoupling protein, a mitochondrial potassium channel, the outer membrane located porin and the permeability transition pore in tumor progression/control. In this review, we focus on mitochondrial channels that have been assigned a definite role in cell death regulation and possess clear oncological relevance. Overall, based on in vivo and in vitro genetic and pharmacological evidence, mitochondrial ion channels are emerging as promising targets for cancer treatment. Keywords: mitochondria; ion channels; direct targeting; pharmacology M ITOCHONDRIA; ION HOM EOSTASIS AND APOPTOSIS; IM PACTS ON SIGNALING AND CANCER The mitochondrial inner membrane (IM M ) surrounding the matrix permits the formation of an electrochemical proton gradient that drives aerobic adenosine triphosphate (ATP) synthesis, and the outer membrane (OM M ) delimits an intermembrane space (IM S) between the OM M and IM M . Proteins with fundamental roles in cell death, such as cytochrome c and SM AC/Diablo, can be released from the IM S upon receipt of a proapoptotic signal and can lead to commitment of the cell to apoptosis. The released serine protease HtrA2/Omi, apoptosis-inducing factor and endo- nuclease G also contribute to the executive phase of apoptosis. 1 M uch effort has been devoted to identifying drugs capable of inducing cancer cell death by stimulating OM M rupture/ permeabilization and release of these proteins. 2 M itochondrial ion channels are therapeutically relevant to cancer because they can influence both OM M and IM M permeability. M embers of the B-cell lymphoma 2 (Bcl-2) family and M AC (mitochondrial apoptosis-induced channel) are directly involved in OM M permeabilization, but they do not function as bona fide ion channels. Excellent reviews are available on their role in this process; 3,4 therefore, the present review focuses on the mitochondrial voltage-dependent anion channel (VDAC, also known as porin) among the OM M channels. Selective induction of IM M permeabilization in cancer cells is also a potential therapeutic strategy because mitochondrial energy production becomes compromised when the IM M becomes freely permeable to solutes, which leads to cell death. As a consequence of changes in ion flux across the IM M , alterations of mitochondrial function occur that produce severe effects on the overall fitness, including ATP production, of these organelles. M ost available data on ion channels or ion flux pathways that modulate cancer cells can be ultimately reconciled with the idea that the perturbation of ion homeostasis within mitochondria leads to cancer cell death via reactive oxygen species (ROS) production, mitochondrial calcium overload and metabolism changes, but other mechanisms of action might also exist. Indeed, mitochondria are crucial for maintaining intracellular Ca 2 þ homeostasis and produce ROS, which are two important factors for cancer cell survival and proliferation. A detailed overview of the above processes is beyond the scope of this review; thus, we briefly describe the general mechanisms by which ion channels may affect ROS production, mitochondrial calcium homeostasis and metabolism. The matrix-negative difference in electrical potential across the IM M (DC m , ranging between À 150 and À 180 mV) is maintained by the proton pumps of the respiratory chain (RC). Consequently, cations (K þ and Ca 2 þ ) flow from the IM S (where the concentra- tion of ions is comparable to cytosolic concentrations because the OM M is permeable to these ions) to the matrix when a permeation pathway opens, as was demonstrated several years ago by experiments with ionophores such as valinomycin. The electrical- equivalent anion outflow is considered much less relevant because the anion pool in the mitochondrial matrix is limited compared with the cytosolic K þ pool. To compensate for charge movement (unless permeating counterions are also present, which is feasible in experimental settings but unusual under physiological conditions), the RC must increase the rate of proton transfer from the matrix to the IM S. To increase RC activity according to the chemiosmotic model, the transmembrane electrochemical proton gradient (D~ m H , composed of mainly DC m ) must decrease. Thus, passive charge flow and D~ m H (DC m ) are coupled, and the opening of K þ or Ca 2 þ channels in the IM M will lead to depolarization. Clearly, this model also functions in reverse; closing cation channels is expected (and observed) to lead to an increase of a D~ m H (DC m ) (that is, hyperpolarization). 1

Department of Biology, University of P adova, P adova, Italy; 2 CNR Institute of Neuroscience and Department of Biomedical Sciences, University of P adova, P adova, Italy and

Department of Molecular Biology, University of Duisburg-Essen, Essen, Germany. Correspondence: P rofessor I Szabo, Department of Biology, University of P adova, Viale G Colombo 3, P adova P D, 35138, Italy. E-mail: [email protected] or [email protected] 3

Such alterations may in turn have consequences on the rate of superoxide formation. 5,6 Hyperpolarization lowers the efficacy of cytochrome c oxidase, 7 induces the reduction of RC complexes and intermediates and thereby increases the probability of a one- electron transfer to oxygen at respiratory complex I. Complex-III- dependent ROS formation can also be significant at high respiration rates (depolarized conditions) because of a high concentration of semiquinone at center ‘O’ (SQ o or Q P ) of the bc 1 complex. 8 Thus, enhanced superoxide (O 2 À ) production occurs in both the matrix and IM S. 7,9–11 In addition, recent data have highlighted that complex II also generates superoxide or hydrogen peroxide molecules at the flavin site II F when electrons are supplied by either succinate or the reduced ubiquinone pool. 12 Irrespective of the site of production, which might depend on the substrates being oxidized, 13 the superoxide anion is the precursor of most ROS. ROS can diffuse to different subcellular compartments. Increased ROS production is important for the maintenance and evolution of the cancerous phenotype. 14 The effects of ROS vary according to the stage of carcinogenesis. In the tumor initiation phase, ROS may cause DNA damage and mutagenesis, whereas in established cancer they may act as signal mediators through the Akt pathway and stimulate proliferation, conferring a growth advantage. 15 ROS may also inhibit Pyruvate Kinase M 2, the embryonic form produced by cancer cells, thereby diverting reducing equivalents to the pentose phosphate pathway. 16 Conversely, ROS release can induce cell death by damaging proteins, lipids and DNA and thus act as anticancer agents. 17–19 In addition, the activation of pro-apoptotic kinases, such as apoptosis signal-regulating kinase 1 (ASK1) and death-associated protein kinases (DAPK; 5 members), 20,21 by ROS is of fundamental importance. 15,17–21 Oxidative stress can also activate the pro- apoptotic Bax and permeability transition pore (M PTP) (see below), 22 whose prolonged opening leads to the loss of the mitochondrial D~ m H , mitochondrial swelling, cristae remodeling and pro-apoptotic factor release. 23 Bax and other Bcl-2 family proteins regulate OM M permeabilization through pore formation and possible regulation of mitochondrial fission and fusion. 24 In light of this dichotomy, both antioxidant and pro-oxidant approaches have been proposed to antagonize cancer. 25–28 Antioxidants are expected to be useful for cancer prevention, whereas pro-oxidants are desirable to potentially and selectively induce cancer cell death. Chronic metabolic oxidative stress, likely occurring in most cancer cell types, can indeed be exploited because these cells exhibit an increased sensitivity to ROS-induced apoptosis. 26 Therefore, therapies that specifically target the RC, either directly or indirectly, to further elevate ROS production should selectively kill cancer cells. Because pharmacological modulation of mitochondrial ion channel function is expected to lead to changes in membrane potential, targeting these channels to increase ROS levels above a critical threshold might be one way to induce cancer cell apoptosis, as detailed below. Furthermore, altered ion fluxes might lead, via hyperpolariza- tion, to an increased driving force for Ca 2 þ entry into the matrix, whereas cation (K þ or M g 2 þ ) channel opening-induced depolar- ization decreases Ca 2 þ uptake. M itochondria have the capacity to accumulate Ca 2 þ , using the mitochondrial calcium uniporter as a highly selective ion channel, 29 to a much higher concentration than found in the cytoplasm. Thus, mitochondria shape cytosolic calcium signals. 30 The mitochondrial matrix possesses a Ca 2 þ buffering system and calcium exit pathways (the Na þ /Ca 2 þ exchanger and transient opening of the permeability transition pore) that normally prevents excessive [Ca 2 þ ] buildup (see, for example, Drago et al. 31 ). However, after apoptotic stimuli, sustained Ca 2 þ release from the endoplasmic reticulum (‘ER stress’) may lead to high mitochondrial matrix Ca 2 þ levels that causes prolonged opening of the permeability transition pore and subsequent loss of mitochondrial D~ m H . Thus, Ca 2 þ overload disrupts energy production, releases proapoptotic proteins into the cytoplasm and enables apoptosis and/or necrosis. 31–33 In addition, mitochondrial Ca 2 þ accumulation also leads to enhanced ROS production through multiple potential mechanisms. 34 Although the exact mechanistic role of mitochondrial Ca 2 þ homeostasis in tumorigenesis (beyond ROS production and modulation of apoptosis) is not well understood, mitochondrial Ca 2 þ is an important signal transducer that can initiate pathways mediating cell death. Therefore, drugs increasing calcium uptake may efficiently kill cancer cells; however, how to selectively target cancer cells instead of healthy cells by inducing mitochondrial calcium accumulation remains unclear. M itochondrial calcium uptake is important for oxidative phosphorylation regulation 35 and energy metabolism control because it enhances the rate of NADH production by modulating the enzymes of the Szent-Gyo ¨ rgyi–Krebs cycle and fatty acid oxidation pathways. 36,37 In general, strategies that increase energy and metabolite production in energy-demanding cancer cells promote a high glycolytic flux rate and thus allow enhanced tumor cell growth. 38,39 Some IM M channels, by regulating DC, matrix volume, matrix acidification and ROS production, would be expected to impact the functionality of mitochondrial RC complexes. Indeed, enhanced substrate oxidation with matrix volume expansion has been demonstrated, 40 and pharmacological activation of calciuminduced potassium channels by NS11021, which affects mitochondrial matrix volume, leads to enhanced respiratory control. 41 Interestingly, the respiration rate and oxidative phosphorylation efficiency were found to be increased in August rats that are characterized by an increased rate of ATPdependent mitochondrial potassium transport as compared with Wistar rats that have lower ATP-dependent potassium channel activity. 42 A recent paper reported the physical interaction and functional coupling of the mitochondrial large- conductance calcium-activated potassium channel (mtBK Ca ) and complex IV of the RC. 43 Whether this physical interaction also occurs in other channels and plays a direct role in the regulation of mitochondrial energy fitness by ion channels is an important and unresolved question. Finally, the changes in matrix volume resulting from channel opening are expected to lead to structural cristae reorganization that has recently been shown to affect respiratory supercomplex organization and respiration efficiency. 44 Along with IM M channels, a role for the OM M channel VDAC in metabolism regulation has become clear; the glycolytic enzyme hexokinase (HK), when bound to VDAC1, regulates metabolite trafficking (including ATP) 45,46 through the OM M channel and provides cancer cells with metabolic advantages. Furthermore, VDAC1-bound HK is less sensitive to inhibition by its product, glucose-6-phosphate, 47 and avoids product inhibition that further boosts a high glycolytic rate. Thus, IM M and OM M channels are expected to affect mitochondrial metabolism and RC function through multiple mechanisms. Therefore, their modulation may lead to cancer cell metabolism alteration, ultimately promoting cell death, as discussed below. In summary, OM M and IM M channel modulation may affect cancer cell fate through different signaling mechanisms. THE IN VITRO AND IN VIVO TARGETING OF M ITOCHONDRIAL ION CHANNELS IN CANCER The mitochondrial channels characterized over the past two decades include the outer membrane-localized VDAC and, in the IM M , potassium channels such as mtK ATP , mtBK Ca , mtIK Ca , mtKv1.3, mtTASK-3, the nonselective permeability transition pore M PTP, chloride channels, the magnesium-permeable M rs-2, the calcium uniporter, uncoupling proteins and proteins that have been shown to function as ion channels in other membranes but not

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yet directly observed in mitochondrial membranes. 48–51 Here, we discuss the in vitro and in vivo evidence for ion channels involved in cancer cell death obtained through either pharmacological or genetic means (Figure 1). Data from animal models clearly demonstrate the importance of mitochondrial channels and their potential as therapeutic cancer targets. The possible roles of individual channels in cell death in relation to the processes discussed above are summarized in Figure 2 and are described in greater detail below. Pharmacological targeting of these channels (summarized in Table 1) and possible pitfalls are also critically discussed. The outer membrane-localized VDAC The major protein of the OM M , VDAC or porin, is required for cancer cell survival and participates in apoptotic signaling. A large body of literature examines the role of VDAC in the regulation of apoptosis. 48,52 VDAC is being studied as a cancer-specific target because tumor cells have increased glycolysis and VDAC expression. 53 The role of VDAC1 and the other isoforms, VDAC2 and VDAC3, in cell death is complex, 54–56 but importantly, in vivo evidence shows that in cancer cells, the association of HK with VDAC1 protects against mitochondrial-mediated apoptosis. Therefore, disruption of the HKVDAC1 complex represents an attractive therapeutic cancer target. Overexpression of hexokinase-1 and 2 (HK2) and their association with VDAC are important characteristics of glycolytic cancers. 57 The expression of VDACs has also been found to be elevated in cancerous cells compared with normal cells and may be altered with chemotherapy. High VDAC levels are an unfavorable prognostic factor; 53 moreover, VDAC downregulation by RNA interference reduces cancer growth. 58 This evidence may seem at odds with the finding that VDAC overexpression induces apoptosis, 59– 61 but it illustrates how the functional meaning of a biological parameter depends on context. VDAC upregulation in cancer goes hand in hand with HK2 upregulation and can be considered a component of glycolytic upregulation. HK2 binding to VDAC,

which allows for ATP transport out of mitochondria, leads to a cancer cell metabolic advantage (termed the Warburg effect), and it antagonizes cell death through the inhibition of Bax-induced cytochrome c release 62,63 and/or inhibition of the mitochondrial permeability transition (M PT). 64 HK dissociation from VDAC seems to favor cell

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death through the disruption of aerobic glycolysis and the cell’s energy balance by altering the interaction of Bcl-2 family proteins with mitochondria and by regulation of ROS production and facilitation of VDAC oligomer formation. 48,55 A class of novel apoptosis-inducing anticancer drugs, which currently comprises small molecules such as clotrimazole, bifonazole (both fungicides, in the tens-of-micromolar range) and methyl jasmonate (M J), act by disrupting the HK-VDAC1 complex (Table 1). Among these small molecules, M J has been demonstrated to be active in preclinical models of melanoma, prostate and breast cancer, lymphoblastic leukemia and multiple myeloma, 65,66 also independently of p53 status and has been tested in at least one pilot human study. 67 However, the major obstacle for adopting jasmonates as anticancer agents is their relatively high dosage needed to exert their action. It should be also noted that different mechanisms of action from the mechanism described above have also been envisioned. M J activates Caspase-3 through ROS production, 68 and another study showed that it also downregulates the expression of proliferating cell nuclear antigen. 69 Furthermore, M J can bind to members of the Aldoketo reductase family 1, 70 which are potential tumor biomarkers involved in steroid metabolism. 71,72 The fact that M J does not affect nontransformed, intact cells is compatible with most suggested mechanisms. Whether M J can also induce cell death in the absence of the proapoptotic Bax/Bak, which occurs in chemoresistant cancer cells, remains unknown. Clotrimazole could have multiple effects because it also acts as a membrane- permeable inhibitor of the KCa3.1 calcium-dependent potassium channel that has also been localized to the IM M 73 and seems to be involved in apoptosis regulation. 74 In addition, peptides corresponding to the amino-terminus of both HK-I 75 and HK-II 62 and a cell-permeable HK-II-based peptide 76 may be potentially useful as pharmacological tools. The HK-VDAC complex (more specifically, HK) can also be targeted by the alkylating agent 3-bromopyruvate (3BP) that has been reported efficacious in in vitro and in vivo studies (for example, see reviews in refs 77–80) and possesses strong potential as an anticancer agent in humans. 79,81 In animal models, 3BP showed high efficacy against advanced-stage malignant tumors by inhibiting both glycolysis and mitochondrial energy generation, possibly by interfering with the HK-VDAC1 complex. 80 Thus, 3BP has been proposed to target metabolism and block energy supplies. In addition, GAPDH, 82 components of the RC, 83 ER and lysosomes are targets of this compound. Its remarkable specificity for cancer cells may be partially attributed to the presence of glycolytic pathway (HK and GAPDH) members among these targets; however, most of 3BP specificity likely stems from facilitated entry into glycolytic cancer cells via the lactate transporter. 84 The transcription factor ATF2, which elicits oncogenic and tumor-suppressor activities in

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melanoma and nonmalignant skin cancer, respectively, has been recently identified as a potent disruptor of the HK1–VDAC1 complex. When localized to mitochondria in a protein kinase C-E- dependent manner, ATF2 increases mitochondrial permeability and promotes apoptosis. 85 The heterocycle erastin has been found to selectively induce the death of cells with mutations in the oncogenes HRas, KRas and BRaf in a VDAC2/3-dependent manner. Apparently, this compound induces oxidative stress involving VDAC through an unknown mechanism. 86 An erastin homolog, PRLX 93936, is currently being evaluated in a clinical trial in multiple myeloma patients (http://www.cancer.gov/clinicaltrials). Finally, the anticancer agent furanonaphthoquinone, isolated from Avicennia plants, induces caspase-dependent apoptosis via ROS production by acting on VDAC. VDAC has also been proposed to mediate exit of superoxide anions from the intermembrane space, and its closure caused internal oxidative stress and sensitized mito- chondria to Ca 2 þ /ROSinduced M PT. 87 M oreover, the anticancer activity of furanonaphthoquinone and ROS production were enhanced by VDAC1 overexpression. 88 VDAC has also been proposed to directly participate in OM M permeabilization and mediate cytochrome c release, either by oligomer formation 48 or by the formation of a large pore comprising VDAC and Bax/Bak (Tsujimoto et al. 89 but see M artinez-Caballero et al. 90 ). Knockdown of VDAC1 prevented cisplatin-induced conformational activation of Bax 91 or selenite- induced cytochrome c release. 92 However, Bax-induced cytochrome c release from mitochondria isolated from wild-type or VDAC1-, VDAC3- and VDAC1/VDAC3-null cells was reported to be identical. 93 In any case, the binding of anti-apoptotic Bcl-2 and Bcl-xL to VDAC1 (with resulting porin activity inhibition) 94 produced anti-apoptotic actions. 95 A recent study reported that constructs consisting of cell-penetrating Antp fused to VDAC1- derived sequences, which prevented the interaction of VDAC1 and HK, Bcl-2 and BclxL, were effective in the selective eradication of B cells from patients with chronic lymphocytic leukemia. 96 In summary, a systematic search for compounds that act on VDAC to antagonize cancer remains to be performed, but those compounds already identified are promising. Inner membrane-localized permeability transition pore A number of cytotoxic agents and cellular stressors trigger the loss of IM M permeability. This process is most often caused by M PT that is considered to be a final common pathway of various forms of cell death. 97,98 In fact, M PT results in a ‘bioenergetic catastrophe’; the transmembrane electrochemical proton gradient dissipates, ATP synthesis ceases and respiratory substrates are lost from the mitochondrial matrix. M PT is caused by the opening of a large, Ca 2 þ -activated and oxidative stress-sensitive pore (M PTP) that creates an IM M permeable to ions and solutes up to 1500 Da molecular weight and leads to matrix swelling. This pore coincides with the mitochondrial megachannel that has been studied by patch clamp and characterized by conductances up to 1.5 nS. 99 M PTP has recently been proposed to be formed by dimers of the FoF1 ATP synthase and Cyclophilin D. 98,100 M PTP/mitochondrial megachannel opening is considered to account for a substantial portion of the tissue damage caused by ischemia/reperfusion and oxidative stress. In cancer cells, signaling pathways are activated that render mitochondria more resistant to M PT induction. 101–103 Because chemotherapeutic agents cause oxidative stress, they may activate signals that induce cell death through M PTP opening. 104 M PT inducers have potential oncological applications, but potential side effects involving the nervous system or cardiac tissues can be expected. A consistent number of compounds (often used at relatively high concentrations) have been shown to induce oxidative stress and/or disruption of Ca 2 þ homeostasis along with M PT. Some of the M PTP-targeting molecules, such as 4-(N-(S-glutathionylacetyl) amino) phenylarsenoxide, are currently being evaluated in clinical trials as promising drugs against refractory tumors. 105,106 M itochondria-penetrating peptides, such as mastoparan-like sequences, peptides of the innate immune system and molecules developed by the Kelley group, 107–109 also induce M PT and could become candidates for future clinical trials. M PTP can also be indirectly activated (or inhibited) through modulatory signaling pathways. For example, in cancer models containing constitutively active extracellular signal-regulated kinase, this kinase acts through the glycogen synthase kinase- 3b/Cyclophilin D axis to repress cell death by M PT inducers such as arachidonic acid or BH3-mimetic EM 20-25. 110 Induction of oxidative stress by the gold complex AUL12 can lead to activation of glycogen synthase kinase-3a/b that favors M PTP opening. 111 A large portion of M PTP-opening inducers (direct or indirect) for which in vivo antitumor activities have been reported 2 are natural compounds, including jasmonates, 112–114 betulinic acid, the synthetic retinoid CD437, 115,116 berberine, 117,118 honokiol, 119,120 a-bisabolol 121 and shikonin 122 (Table 1). Among these com- pounds, betulinic acid is currently in a phase I /II clinical trial for dysplastic nevi (http://www.cancer.gov/clinicaltrials). A retinoid analog, NRX 195183, is also in a phase II trial for acute promyelocytic leukemia (http://www.cancer.gov/clinicaltrials). A specific, powerful M PTP inhibitor is also available: cyclosporin A, a cyclic endecapeptide. 123–125 Cyclosporin A inhibits the M PTP through binding to matrix cyclophilin, a peptidyl-prolyl cis–trans isomerase, and acts as an immunosuppressant by inhibiting calcineurin. Further evidence for the link between M PTP and cancer is illustrated by the observation that patients treated with cyclosporin A to prevent rejection of organ transplants have a high incidence of cancer. 126 The IM M potassium channel Kv1.3 As mentioned above, IM M -localized potassium channels are predicted to participate in the regulation of mitochondrial membrane potential, volume and ROS production. Our research groups have identified a potassium channel, mtKv1.3, in the IM M of several cell types and have shown with mitochondria from cells expressing or lacking this channel that mtKv1.3 activity indeed has an impact on DC m and on ROS production. 127,128 Similar to some other IM M channels, 50 mtKv1.3 is the mitochondrial counterpart of the plasma membrane-localized Shaker family potassium channel, Kv1.3. Its crucial role in apoptosis became evident because the expression of a mitochondria-targeted Kv1.3 construct was sufficient to induce cell death upon apoptotic stimuli in CTLL-2 T lymphocytes that lack Kv channels and are otherwise resistant to apoptosis. A physical interaction between Bax and mtKv1.3 has been demonstrated in apoptotic cells, and Bax has been shown to inhibit channel activity at nM concentrations. 129,130 Incubation of Kv1.3-positive isolated mitochondria with Bax or specific mtKv1.3 inhibitors triggered apoptotic events, including membrane potential changes (hyperpolarization followed by depolarization because of M PTP opening), ROS production and cytochrome c release; however, Kv1.3-deficient mitochondria were resistant to these inhibitors. The highly conserved Bax lysine residue 128 was shown to protrude into the intermembrane space. 131 This residue is responsible for the inhibitory effect of Bax on Kv1.3 activity because it mimics a crucial lysine residue in Kv1.3blocking peptide toxins. Indeed, mutant BaxK128E did not exert its effects on Kv1.3 and mitochondria, and it did not mediate apoptosis in Bax/Bak double-knockout mouse embryonic fibroblasts, indicating that the toxin-like action of Bax on Kv1.3 triggers the above described mitochondrial events. 130 Three membrane-permeable inhibitors of Kv1.3, Psora-4, PAP-1 and clofazimine, are able to induce cell death at mM concentra- tions by directly targeting mtKv1.3 in different cancer cell lines, in

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contrast to the membrane-impermeable high-affinity Kv1.3 inhibitors ShK and margatoxin. 132,133 Genetic deficiency or small interfering RNA-mediated downregulation of Kv1.3 abrogated the effects of the membrane-permeable drugs that also killed cells in the absence of Bax and Bak, in agreement with the above described model (Figure 3). In vivo, intraperitoneal injection of clofazimine in an orthotopic melanoma B16F10 mouse model reduced tumor size by 90%, without causing obvious side effects. 133 Recently, promising results were obtained with primary human cancer cells from patients with chronic lymphocytic leukemia, where drugs significantly and exclusively decreased the survival of pathologic B cells (which express higher levels of Kv1.3 compared with B cells from normal subjects) by inducing the crossing of a critical ROS threshold. 134 Thus, the selective apoptosis-inducing action of these drugs on tumor cells seems to be related to a synergistic effect of an altered cancer cell redox state and higher Kv1.3 expression. 134,135 Interestingly, As 2 O 3 , a clinically active antileukemic agent, has been reported to inhibit mitochondrial respiratory function, increase ROS generation and enhance the activity of other O 2 . À -producing agents against cultured and primary patient leukemia cells. 136 Because clofazimine is already used clinically to treat some autoimmune diseases and leprosy 137 and shows an excellent safety profile, targeting mtKv1.3 is a feasible cancer therapy for at least some cancer types. Interestingly, altered Kv1.3 expression is observed in several different cancer cell lines and tumors, 138 and a correlation between Kv1.3 expression and cell sensitivity to clofazimine has been reported. 139 In addition, the ability of these drugs to induce cell death in the absence of Bax and Bak, similar to other agents, 140–142 could be useful because downregulation of these pro-apoptotic proteins represents a common tumor cell resistance mechanism. 143–146 Calcium-dependent potassium channels The activity of an intermediate conductance potassium channel (IK mitochondrial

Ca

, KCa3.1) has been recorded from the inner

membranes of human cancer cells. 73,147 . IK Ca can be selectively inhibited by low concentrations of clotrimazole and TRAM -34. This latter drug has been reported to synergistically increase the sensitivity of melanoma cells to the death receptor ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) via its action on mtIK Ca . 74 However, the effects of TRAM -34 on mitochondrial bioenergetics have not yet been investigated in detail, but TRAM -34 has been reported to induce hyper- polarization of the mitochondrial membrane (which is expected if an influx of positively charged ions/molecules is inhibited), confirming that functional IK Ca is expressed in the IM M . Interestingly, TRAM -34 application induced Bax translocation to the mitochondria, representing an early step of apoptosis. Both TRAIL and TRAM -34 are characterized by a relatively good safety profile, suggesting that co-administration of these two drugs might be exploited to treat melanoma. In addition to the intermediate conductance channels, the presence of small-conductance calciumactivated potassium (SK2/ KCa2.2) channels in the IM M has been recently reported. 148,149 Its pharmacological activation in a neuronal cell line exposed to toxic glutamate levels attenuated the loss of the mitochondrial transmembrane potential, blocked mitochondrial fission, prevented the release of proapoptotic mitochondrial proteins and reduced cell death. 149 SK2 channel opening prevented mitochondrial calcium overload and mitochondrial superoxide formation. It will be interesting to investigate whether the application of mtSK2 inhibitors alone or in combination with other chemotherapeutics could promote cell death in cancer cells expressing SK2. To our knowledge, whether the mitochondrial large-conduc- tance Ca 2 þ -activated K þ channel (BK Ca /KCa1.1) 150 has a role in cancer has not been investigated. However, the channel opener CGS7184 has been reported to induce the glioma cell death that was accompanied by increased respiration and mitochondrial depolarization, 151 perhaps downstream of an increased Ca 2 þ release from the ER. 152

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TWIK-related acid-sensitive K þ channel-3 (TASK-3) A protein recognized by an antibody against TASK-3 (TWIK-related acid-sensitive K þ channel-3; KCNK9), a two-pore potassium channel, was identified in the mitochondria of melanoma, keratinocytes 153 and healthy intestinal epithelial cells. 154 The reduction of TASK-3 expression in WM 35 melanoma cells compromised mitochondrial function and cell survival. 155 TASK-3 knockdown also decreased human keratinocyte HaCaT cell viability following ultraviolet B irradiation. 156 In mitochondria isolated from HaCaT cells, TASK-3-compatible activity has been recorded for the first time by electrophysiology (patch clamp). 156 Two aspects of TASK-3 still require investigation: first, whether the observed migration and invasion-reducing effects of TASK-3 overexpression in breast cancer cells 157 and increased apoptosis induced by TASK-3 blockers (zinc and methanandamide) in ovarian carcinoma 158 are correlated with the mitochondrial channel expression and, second, whether pharmacological action on mtTASK-3 can affect cancer cell death. However, because no highly specific modulators are available, this task remains difficult. Uncoupling protein (UCP) Uncoupling proteins (UCP1–5) belong to the superfamily of mitochondrial anion-carrier proteins, and UCP1 has recently been shown to mediate fatty acid-regulated proton transport. 159 The widely expressed UCP-2 has also been proposed to transport protons, 160 (for review, see Cannon and Nedergaard 161 ), although its physiological importance and the occurrence of UCP2- mediated proton leak are not entirely clear and remain debatable. UCP2 has been suggested to regulate cell survival by decreasing mitochondrial ROS because it might limit the maximal value of the proton gradient across the IM M , and a depolarizing proton leak would be expected to diminish superoxide production 162,163 (see, for example, Cannon et al. 164 for a discussion on this topic). Increased ROS production was observed in UCP2 knockout mice, whereas UCP2 overexpression may contribute to a higher apoptotic threshold promoting cancer cell survival because it prevents oxidative injury. Indeed, UCP2 overexpression, documented in numerous tumor types, including leukemia, ovarian, bladder, esophageal, testicular, colorectal, kidney, pancreatic, lung and prostate tumors, has been shown to protect cells from oxidative stress 165 and even to abolish the apoptosis-inducing effects of chemotherapeutic drugs. 166 Interestingly, UCP2 overexpression has also been proposed to directly contribute to the Warburg phenotype 167 and to the development of tumors in an orthotopic in vivo model of breast cancer. 168 In support of this hypothesis, UCP2 expression in M CF7 breast cancer cells was shown to lead to a decreased mitochondrial membrane potential and increased tumorigenic properties. These studies suggest that UCP2 overexpression is involved in the development of a variety of cancers and that UCP2 can be considered as a promising oncological target, although the actual ions transported by UCP2 under physiological conditions are still uncertain. As recently suggested, 162 UCP2 may act as an uniporter for pyruvate, presumably promoting pyruvate efflux from the matrix and thus restricting glucose availability for mitochondrial respiration 162 and promoting mitochondrial fatty acid oxidation. Efflux of pyruvate would aid highly glycolytic cells, in which large amounts of pyruvate would otherwise put enormous redox pressure on the mitochondria. Nevertheless, a plant-derived small molecule, genipin, has been identified as an agent capable of suppressing the tumor-promoting functions of UCP2 presumably by abolishing UCP2-mediated proton leak. 168 In sharp contrast with the above studies, when highly metastatic M DA-M B-231 breast cancer cells recombinantly overexpressing UCP1, UCP2 or UCP3 were injected into the flanks of athymic nude mice, in vivo tumor growth was reduced 2.2-fold compared with empty-vector control cells. 169 The authors interpreted their results as a consequence of UCP-induced increased autophagy and decreased mitochondrial function. This unexpected observation warrants further investigation. M itochondrial calcium uniporter M CU The mitochondrial Ca 2 þ ‘uniporter’ (M CU), a calcium-selective ion channel, 29 is responsible for low-affinity calcium uptake into the mitochondrial matrix. The recent identification of the protein responsible for this activity 170,171 has produced new perspectives pertaining to the control of Ca 2 þ signaling in the cell. In fact, M CU may be a very useful tool to influence a number of cellular calcium-dependent processes, including cell death. 51 For example, subthreshold apoptotic stimulation in synergy with cytosolic Ca 2 þ waves induced M PTP opening 172 and, moreover, M CU overexpression resulted in increased apoptosis upon with H 2 O 2 and C2-ceramide challenges. 170 In addition, recent data suggest that in the absence of M ICU1, a proposed negative regulator of M CU, mitochondria become constitutively loaded with Ca 2 þ , resulting in excessive ROS generation and sensitivity to apoptotic stress. 173 To avoid calcium overload, M CU activity is also regulated by a dominant-negative subunit, M CUb. 174 As for a possible relationship between M CU and cancer, the M CU-targeting microRNA miR-25 has been shown to affect Ca 2 þ homeostasis in colon cancer cells through the specific downregulation of M CU. The miR-25 caused a strong decrease in mitochondrial Ca 2 þ uptake and, importantly, conferred resistance to Ca 2 þ -dependent apoptotic challenges. 175 Thus, M CU could be an important protein for tumorigenesis, and its specific pharmacological activators, if identified, might become useful tools. However, the situation is likely more complex. M CU overexpression has been observed in breast cancer samples and has been suggested to provide a survival advantage against some cell death pathways. 176 Pharmacological or small interfering RNA-mediated inhibition of mitochondrial Ca 2 þ entry sensitized cancer cells to positively charged gold nanoparticles through the induction of endoplasmic reticulum stress. 177 Further complications arose from recent work reporting that epidermal growth-induced epithelial– mesenchymal transition in breast cancer cells was not associated with changes in M CU expression, as assessed by quantitative reverse transcriptase–PCR. 178 Finally, in sharp contrast with the extensive literature suggesting that alterations in mitochondrial calcium play a central role in acute metabolic regulation and determination of a threshold for cell death, the basal metabolism of knockout animals was not markedly altered in the absence of M CU. 179 Furthermore, although experiments with M CU À / À mitochondria confirmed that M CU is required for calcium- induced M PTP opening, M CU À / À mice exhibited no evidence of protection from ischemia–reperfusion-mediated injury. In this study, tumor incidence in knockout mice was not assessed. The forthcoming elucidation of M CU regulation, 180–182 as well as the creation of a conditional knockout mouse, might provide explanation for these apparently contrasting findings. Acid-sensing ion channel 1a In a recent study, the sodium-permeable acid-sensing ion channel 1a (ASIC1a) was documented in mitochondria, although the transport properties of this mitochondrial protein were not defined. 183 Interestingly, neurons from ASIC1a À / À mice were resistant to cytochrome c release and inner mitochondrial membrane depolarization, and mitochondria from these cells displayed an enhanced Ca 2 þ retention capacity and oxidative stress response. The authors proposed that mitochondrial ASIC1a may serve as a regulator of M PTP through a still ill-defined mechanism, thus contributing to oxidative neuronal cell death. Interestingly, lack of ASIC1 prevented axonal degeneration in experimental autoimmune encephalomyelitis; however, M PTP

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THERAPEUTIC PERSPECTIVES The concept that mitochondria can serve as promising pharma- cological targets in oncology is gaining increasing support. One intervention strategy envisions direct targeting of mitochondrial channels to induce the death of unwanted, cancerous cells. Although much work remains in this relatively new field, studies involving VDAC, inner membrane K þ and Ca 2 þ channels and the permeability transition pore justify high expectations. To produce advances in this field, further progress should focus on under- standing the functions of these and other channels in normal cells, mechanisms involved in cell death induction upon their manip- ulation, characterization of other mitochondrial channels (for example, the 107-pS anion channel) in an oncological context, screening of potential drugs and improvements specifically targeting drugs to mitochondria and to cancerous cells. Table 1 summarizes the currently available pharmacological data target- ing mitochondrial ion channels obtained in various cancer cells. To expand this field, studies should address the expression level of a set of ion channels in cancer tissues. For example, among the channels that are also present in the mitochondrial membranes, IK Ca (KCa3.1) expression has been shown to be significantly increased in breast tumors bearing a p53 mutation, and a correlation was detected between high expression of this channel and high tumor grades. 185 The same situation occurs for VDAC1, VDAC2 and VDAC3. 185 Conversely, BK Ca (KCa1.1) expression was reduced in patients with higher grade tumors compared with patients with lower grade tumors. 185 Emerging data on mitochondrial ion channels might help to identify specific drugs that can be used to obtain maximal cancer-killing efficacy. The major hurdle from a pharmacological perspective concerns the specificity and membrane permeability of drugs acting on mitochondrial channels. In many cases, the used inhibitors have multiple off-target effects as well (for example, methyl jasmonate, honokiol). Because several channels are present in both the plasma membrane and the mitochondrial membrane, drugs targeting the mitochondrial channels might exert undesired effects on processes linked to plasma membrane channels of healthy cells (for example, proliferation). Furthermore, only cell membrane-permeable drugs will reach mitochondrial channels, and a portion of these drugs is likely to be retained in cellular membranes. Therefore, higher drug concentrations than those concentrations sufficient to block the channels are needed to exert an effect on cell viability, and these concentrations may lead to a loss of specificity. Different strategies can be envisioned to create more efficient targeting of mitochondrial channels. Cell- penetrating peptides (such as the VDAC-related peptides) are one possibility, but clinical problems could arise, as these peptides are able to cross the blood–brain barrier. Another strategy might be derivatization of the drugs by adding a permeating positively charged moiety 186–188 that is able to drive accumulation of the drugs in mitochondria. In summary, to exploit mitochondrial channels as oncological targets, we should explore exactly how these ion channels contribute to cell fate, and state-of-the-art pharmacological tools should be developed and used for specific targeting. CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEM ENTS We are grateful to all co-authors for their important contributions to the work carried out in their laboratories and reported in this review. We apologize for not citing all publications that have been published on this topic. The work carried out in the authors’ laboratories was supported in part by grants from the Italian Association for Cancer Research (AIRC; Grant 11814 to IS), the EMBO Young Investigator P rogram grant (to IS), the P rogetti di Rilevante Interesse Nazionale (P RIN) program (2010CSJX4F to IS and 20107Z8XBW_004 to MZ), the Fondazione Cassa di Risparmio di P adova e Rovigo (to MZ), the CNR P roject of Special Interest on Aging (to MZ), the DFG Grant Gu 335/13–3 (to EG), the International Association for Cancer Research (to EG) and the P rogetto Giovani Studiosi 2012 (to LL).

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